Car-expressing NK-92 cells as cell therapeutic agents

ABSTRACT

The present invention relates to an ErbB2-specific NK-92 cell or cell line containing a lentiviral vector encoding a chimeric antigen receptor and preferably two vector integration loci in its cellular genome. The present invention further relates to the use of the ErbB2-specific NK-92 cell or cell line in the prevention and/or treatment of cancer, preferably ErbB2-expressing cancers. The present invention further relates to the use of the ErbB2-specific NK-92 cell or cell line as targeted cell therapeutic agent and/or for adoptive cancer immunotherapy. The present invention further relates to a method for generating an ErbB2-specific NK-92 cell or cell line as well as to a method for identifying an ErbB2-specific NK-92 cell or cell line and to the ErbB2-specific NK-92 cell or cell line obtained or identified by the methods as well as their uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/319,660, filedDec. 16, 2016, which is a 35 USC § 371 national stage entry ofInternational Application No. PCT/EP2015/063674 filed Jun. 18, 2015,which claims the benefit of European Patent Application No. 14173020.0filed Jun. 18, 2014, each of which are incorporated by reference hereinin their entireties.

The present invention relates to an ErbB2-specific NK-92 cell or cellline containing a lentiviral vector encoding a chimeric antigen receptorand preferably two vector integration loci in its cellular genome. Thepresent invention further relates to the use of the ErbB2-specific NK-92cell or cell line in the prevention and/or treatment of cancer,preferably ErbB2-expressing cancers. The present invention furtherrelates to the use of the ErbB2-specific NK-92 cell or cell line astargeted cell therapeutic agent and/or for adoptive cancerimmunotherapy. The present invention further relates to a method forgenerating an ErbB2-specific NK-92 cell or cell line as well as to amethod for identifying an ErbB2-specific NK-92 cell or cell line and tothe ErbB2-specific NK-92 cell or cell line obtained or identified by themethods as well as their uses.

BACKGROUND OF THE INVENTION

Natural killer (NK) cells are an important effector cell type foradoptive cancer immunotherapy. Similar to T cells, NK cells can bemodified to express chimeric antigen receptors (CARs) to enhanceantitumor activity.

Successful application of CAR-modified T cells in patients withCD19-positive malignancies has demonstrated the potency of this approachfor adoptive cancer immunotherapy (see e.g. Grupp et al., 2013), and CART cells targeting a variety of different tumor antigens are under activeclinical development (Kalos et al., 2013). CAR-mediated retargeting ofnatural killer (NK) cells has been attempted less frequently, and so farno clinical data for such an approach are available. NK cells play animportant role in cancer immunosurveillance, and represent an importanteffector cell type for adoptive cancer immunotherapy (Geller and Miller,2011). In contrast to T cells, they do not require prior sensitizationand recognition of peptide antigens presented in complex with MHCmolecules. Instead, their cytotoxicity can be triggered rapidly uponappropriate stimulation through germline-encoded cell surface receptors(Koch et al., 2013), that in part signal through CD3ζ. Hence,CD3ζ-containing CARs readily link to endogenous signaling pathways in NKcells and trigger cytolytic activity (see e.g. Müller et al., 2008).Despite these advances, experience with CAR-engineered NK cells andtheir clinical development is still limited. Due to efficient antiviraldefense mechanisms, gene transfer into NK cells with retro- andlentiviral vectors as well as physical transfection methods are lessefficient than in T cells, complicating the generation of large numbersof CAR-expressing cells (Boissel et al., 2009). This restriction can beovercome by employing clinically applicable NK cell lines such as NK-92,which allow isolation and expansion of CAR-expressing cells from a bulkof untransduced cells (Müller et al., 2008).

Phase I studies in cancer patients demonstrated the safety of infusionof unmodified NK-92 cells, which were irradiated prior to application toprevent permanent engraftment. Thereby clinical signs of activity wereonly observed in a subset of patients (Tonn et al., 2013), likely due toinsufficient tumor recognition by the unmodified NK-92 cells which lacka tumor-specific receptor.

The present invention aims to provide improved CAR-engineered NK cells,which are suitable for clinical use, in particular in the treatment ofcancers and as targeted cell therapeutic agents.

It is a further objective of the present invention to provide means andmethods for generating and molecularly identifying improvedCAR-engineered NK cells.

SUMMARY OF THE INVENTION

According to the present invention this object is solved by anErbB2-specific NK-92 cell or cell line, containing a lentiviral vectorencoding a chimeric antigen receptor (CAR) comprising

-   -   an ErbB2-specific scFv antibody fragment, a hinge region,        transmembrane and intracellular domains of CD28, and        intracellular domain of CD3 zeta,        wherein said vector is genomically integrated (i), preferably in        an intergenic region on chromosome 2, and (ii) in the TRAF2 gene        on chromosome 9.

According to the present invention this object is solved by providingthe NK-92 cell or cell line according to the invention for use inmedicine.

According to the present invention this object is solved by providingthe NK-92 cell or cell line according to the invention for use in theprevention and/or treatment of cancer, preferably ErbB2-expressingcancers.

According to the present invention this object is solved by providingthe NK-92 cell or cell line according to the invention for use astargeted cell therapeutic agent and/or for adoptive cancerimmunotherapy.

According to the present invention this object is solved by a method forgenerating an ErbB2-specific NK-92 cell or cell line, comprising thesteps of:

(1) providing a vector for transducing NK-92 cells,

(2) transducing NK-92 cells with said vector,

(3) deriving/generating single cell clones by limiting dilution;

(4) identifying CAR-expressing cells by flow cytometric analysis withErbB2-Fc fusion protein,

(5) selecting cell clone(s) which display high and stable CAR-expressionduring continuous culture,

(6) evaluating cytotoxic activity of the retargeted cells againstErbB2-expressing cells,

(7) evaluating cytotoxic activity of the retargeted cells againstErbB2-negative cells,

(8) selecting cell clone(s) which display high cytotoxicity againstErbB2-expressing cells and low or no cytotoxicity against ErbB2-negativecells, and

(9) determining number and position of vector integration, and selectingthe cell clones exhibiting vector intergration in an intergenic regionon chromosome 2 and in the TRAF2 gene on chromosome 9.

According to the present invention this object is solved by a method foridentifying an ErbB2-specific NK-92 cell or cell line, comprising thesteps of:

-   -   determining number and position of vector integration in cell        (clones), and selecting the cell (clones) exhibiting vector        integration in an intergenic region on chromosome 2 and in the        TRAF2 gene on chromosome 9.

According to the present invention this object is solved by an NK-92cell or cell line obtained or identified by the methods according to theinvention.

According to the present invention this object is solved by providingthe NK-92 cell or cell line obtained or identified by the methodsaccording to the invention for use in the prevention and/or treatment ofcancer, preferably ErbB2-expressing cancers.

According to the present invention this object is solved by providingthe NK-92 cell or cell line obtained or identified by the methodsaccording to the invention for use as targeted cell therapeutic agentand/or for adoptive cancer immunotherapy.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Before the present invention is described in more detail below, it is tobe understood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art. For the purpose of thepresent invention, all references cited herein are incorporated byreference in their entireties.

Clinically Usable CAR-Expressing Human NK-92 Cells

As described above, the present invention provides an ErbB2-specificNK-92 cell or cell line,

The ErbB2-specific NK-92 cell or cell line of the present inventioncontains

-   -   a lentiviral vector encoding a chimeric antigen receptor        comprising        -   an ErbB2-specific scFv antibody fragment, a hinge region,            transmembrane and intracellular domains of CD28, and            intracellular domain of CD3 zeta.

The ErbB2-specific NK-92 cell or cell line preferably contains twovector integration loci in its cellular genome.

The ErbB2-specific NK-92 cell or cell line is characterized in that saidvector is genomically integrated (i), preferably in an intergenic regionon chromosome 2, and (ii) in the TRAF2 gene on chromosome 9.

The unmodified NK-92 cell or cell line is known in the art. See e.g.Gong et al., 1994 or WO 98/49268 A1. NK-92 cells are deposited with theAmerican Type Tissue Collection (ATCC no. CRL-2407).

Vector Integration

The ErbB2-specific NK-92 cell or cell line can be molecularlycharacterized and identified by said two vector integrations.

Preferably, the NK-92 cell or cell line of the present invention ischaracterized in that by PCR analysis of the genomic DNA of said cell orcell line at least one of the following amplification products is/areobtained:

-   -   PCR with primers of SEQ ID NOs. 1 and 2 yields an amplification        product with the nucleotide sequence of SEQ ID NO. 9;    -   PCR with primers of SEQ ID NOs. 3 and 4 yields an amplification        product with the nucleotide sequence of SEQ ID NO. 10;    -   PCR with primers of SEQ ID NOs. 5 and 6 yields an amplification        product with the nucleotide sequence of SEQ ID NO. 11;    -   PCR with primers of SEQ ID NOs. 7 and 8 yields an amplification        product with the nucleotide sequence of SEQ ID NO. 12.

PCR analysis of genomic DNA of the cell or cell line of the inventionwith oligonucleotide primers hybridizing to genomic and vector DNAsequences adjacent to 5′ and 3′ junction sites of chromosomal andintegrated vector sequences (namely the oligonucleotide primersspecified in SEQ ID NO. 1 and 2, 3 and 4, 5 and 6, 7 and 8) yieldamplification products of defined length and sequence (namely the PCRfragments specified in SEQ ID NO. 9, 10, 11, 12), confirming the vectorintegrations and molecularly identifying the cell (clone) NK-92 of thepresent invention.

1) Oligonucleotide primers hybridizing to genomic and vector DNAsequences adjacent to 5′ and 3′ junction sites of chromosomal andintegrated vector sequences:

a) CAR vector integration in TRAF2 gene

a-1) 5′ part of vector integration

Forward PCR primer TRAF2-F1:

[SEQ ID NO. 1] CTTCAGCAGGGACCAGAAACAAReverse PCR primer CAR-R1 (vector sequence underlined)

[SEQ ID NO. 2] CCGCTTAATACTGACGCTCTCGa-2) 3′ part of vector integrationForward PCR primer CAR-F1 (vector sequence underlined)

[SEQ ID NO. 3] ATCGCCACGGCAGAACTCAReverse PCR primer TRAF2-R1

[SEQ ID NO. 4] GACCCTTCACCCAACGCTTAGb) CAR vector integration in intergenic region of chromosome 2b-1) 5′ part of vector integrationForward PCR primer IGCHR2-F1

[SEQ ID NO. 5] TCAGTGGAATGGGCAGCTTCAAGTReverse PCR primer CAR-R2 (vector sequence underlined)

[SEQ ID NO. 6] TTCAGCAAGCCGAGTCCTGCGTb-2) 3′ part of vector integrationForward PCR primer CAR-F2 (vector sequence underlined)

[SEQ ID NO. 7] ACTGATAATTCCGTGGTGTTGTReverse PCR primer IGCHR2_CAR-R1 (vector sequence underlined)

[SEQ ID NO. 8] CACTGTGGCTCACTGCTAGA2) Amplification products of defined length and sequence:a) Amplification product of CAR vector integration in TRAF2 geneCAR vector integration in TRAF2 gene5′ part of vector integrationPCR product TRAF2-CAR (5′) from genomic DNA of NK-92/5-28.z cellsPrimers: TRAF2-F1 (SEQ ID NO. 1), CAR-R1 (SEQ ID NO. 2)lower case letters: TRAF2 geneupper case letters: vector sequenceLength: 587 nucleotides

SEQ ID NO. 9 cttcagcagggaccagaaacaaaactcacactctttcttctctgagttga  50gactggaaaaatgaaagattgttttaggggaaacttgagggaacagtctg 100ggcagcctgcagggcatggccctgttcctccagggctgggaaagtcagca 150ctgctttctggtggcgaACTGGAAGGGCTAATTCACTCCCAACGAAGACA 200AGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAG 250CCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAA 300AGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTC 350TGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTA 400GCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGC 450TCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAG 500GGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAA 550GGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGG 587b) Amplification product of CAR vector integration in TRAF2 geneCAR vector integration in TRAF2 gene3′ part of vector integrationPCR product CAR-TRAF2 (3′) from genomic DNA of NK-92/5-28.z cellsPrimers: CAR-F1 (SEQ ID NO. 3), TRAF2-R1 (SEQ ID NO. 4)lower case letters: TRAF2 geneupper case letters: vector sequenceLength: 503 nucleotides

SEQ ID NO. 10 ATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGG  50GGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGAATTCGATAC 100TCGAGGTCGAGGCAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACA 150AGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAA 200GGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGG 250GTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAG 300GGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTA 350GTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACC 400CTTTTAGTCAGTGTGGAAAATCTCTAGCAccttccctctgcagctgctgg 450ctcagccgattgtatatgctgggagctctgcactaagcgttgggtgaagg 500 gtc 503c) Amplification product of CAR vector integration in intergenic regionof chromosome 2CAR vector integration in intergenic region of chromosome 25′ part of vector integrationPCR product IGCHR2-CAR (5′) from genomic DNA of NK-92/5-28.z cellsPrimers: IGCHR2-F1 (SEQ ID NO. 5), CAR-R2 (SEQ ID NO. 6)lower case letters: intergenic region chromosome 2upper case letters: vector sequenceLength: 679 nucleotides

SEQ ID NO. 11 tcagtggaatgggcagcttcaagttgatgtcatttcaatagtaacttatt  50tcagtctacatacttcccaagaatgcaccatctcttttttatgtatttat 100tattttgagaaagagtctcactctgtcgcccaggctggagtgcaatggca 150tgatcttggctcactgtaacctccgtctcctgggttcaagccattctcct 200gtctcagcctcccgggtagtggggttataggcacacaccaccacgcccgg 250ctaatttttgtatttttagtaaagatggggtttcaccatgttggccaggc 300tgggctcaaactcttgacttcaggtgatccgcccaccttggcctcccaaa 350gtgctgggatgacaggcACTGGAAGGGCTAATTCACTCCCAACGAAGACA 400AGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAG 450CCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAA 500AGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTC 550TGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTA 600GCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGC 650TCTCTCGACGCAGGACTCGGCTTGCTGAA 679d) Amplification product of CAR vector integration in intergenic regionof chromosome 2CAR vector integration in intergenic region of chromosome 23′ part of vector integrationPCR product CAR-IGCHR2 (3′) from genomic DNA of NK-92/5-28.z cellsPrimers: CAR-F2 (SEQ ID NO. 7), IGCHR2_CAR-R1 (SEQ ID NO. 8)lower case letters: intergenic region chromosome 2upper case letters: vector sequenceLength: 376 nucleotides

SEQ ID NO. 12 ACTGATAATTCCGTGGTGTTGTCGAATTCGATACTCGAGGTCGAGGCAAT  50TCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCT 100TAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCC 150AACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGAC 200CAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAA 250GCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGT 300TGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGG 350AAAATCTCTAGCAgtgagccacagtg 376

Reduced Natural Cytotoxicity and High Specific Cytotoxicity

Preferably, the NK-92 cell or cell line of the present invention ischaracterized in that:

-   -   the NK-92 cell or cell line shows reduced or no natural        cytotoxicity, i.e. reduced or no cytotoxicity to ErbB2-negative        cells which are lysed by unmodified NK-92 cells,        and/or    -   the NK-92 cell or cell line shows increased specific        cytotoxicity, i.e. increased cytotoxicity to ErbB2-expressing        tumor cells compared to unmodified NK-92 cells.

Said reduced natural cytotoxicity constitutes an important safetyfeature of the cell or cell line of the invention, in particular in viewof a clinical use.

The NK-92 cell or cell line of the present invention—in contrast tounmodified NK-92 cells—lyse ErbB2-expressing tumor cells with highefficiency, but are less likely than unmodified NK-92 cells to attackErbB2-negative non-target cells.

Further Features

In one embodiment, the ErbB2-specific scFv antibody fragment comprisesor consists of the amino acid sequence of SEQ ID NO. 13 (scFv FRP5)and/or is encoded by the nucleotide sequence of SEQ ID NO. 14.

ErbB2-specific scFv FRP5 is further described in EP 2 164 516 B1 andU.S. Pat. No. 7,887,801 B2.

Chimeric antigen receptors (CARs) are known in the art. TheErbB2-specific CAR used in the present invention comprises:

(i) an ErbB2-specific scFv antibody fragment,

(ii) a hinge region,

(iii) transmembrane and intracellular domains of CD28, and intracellulardomain of CD3 zeta.

In one embodiment, the chimeric antigen receptor (CAR) comprises orconsists of the amino acid sequence of SEQ ID NO. 15 and/or is encodedby the nucleotide sequence of SEQ ID NO. 16.

Suitable CARs are further described in WO 2012/031744.

For example, the lentiviral transfer plasmid pS-5.28.z-W encoding underthe transcriptional control of the Spleen Focus Forming Virus promoter(SFFV) the chimeric antigen receptor (CAR) consisting of animmunoglobulin heavy chain signal peptide, the ErbB2-specific scFv(FRP5) antibody fragment, a hinge region (CD8α), followed bytransmembrane and intracellular domains of CD28 and the intracellulardomain of CD3ζ. The CAR-encoding sequence is flanked by 5′ and 3′ longterminal repeats (LTR) of the vector.

Natural killer (NK) cells are an important effector cell type foradoptive cancer immunotherapy. Similar to T cells, NK cells can bemodified to express chimeric antigen receptors (CARs) to enhanceantitumor activity, but experience with CAR-engineered NK cells islimited and data on clinical development are lacking. Here, theinventors redirected clinically usable human NK-92 cells to thetumor-associated ErbB2 (HER2) antigen expressed at elevated levels bymany cancers of epithelial origin.

Following GMP-compliant procedures, the inventors generated anNK-92/5.28.z single cell clone expressing an ErbB2-specific CAR withCD28 and CD3ζ signaling domains.

Vector integrations in the NK-92/5.28.z cell clone were mapped by linearamplification-mediated PCR and DNA sequencing following establishedprocedures (Schmidt et al., 2003), which revealed one vector integrationeach in an intergenic region on chromosome 2, and in the TRAF2 gene onchromosome 9. As discussed above, PCR analysis of genomic DNA witholigonucleotide primers hybridizing to genomic and vector DNA sequencesadjacent to 5′ and 3′ junction sites of chromosomal and integratedvector sequences (e.g. the oligonucleotide primers specified in SEQ IDNO. 1 and 2, 3 and 4, 5 and 6, 7 and 8) yield amplification products ofdefined length and sequence (e.g. the PCR fragments specified in SEQ IDNO. 9, 10, 11, 12), confirming the vector integrations and molecularlyidentifying the cell clone NK-92/5.28.z.

NK-92/5.28.z cells efficiently lysed ErbB2-expressing and otherwiseNK-resistant tumor cells in vitro, but did not lyse ErbB2-negative cellsresistant to unmodified NK-92. Unexpectedly, NK-92/5.28.z cells alsodisplayed reduced cytotoxicity to ErbB2-negative cells which are readilylysed by unmodified NK-92 cells. This constitutes an important safetyfeature of NK-92/5.28.z cells which in contrast to unmodified NK-92cells lyse ErbB2-expressing tumor cells with high efficiency, but areless likely than unmodified NK-92 cells to attack ErbB2-negativenon-target cells. Specific recognition of tumor cells and antitumoractivity were retained in vivo, resulting in homing of NK-92/5.28.zcells to orthotopic breast carcinoma xenografts and reduction ofpulmonary metastasis of renal cell carcinoma cells. γ-irradiation as apotential safety measure for clinical application prevented replicationof the cells, while in vitro and in vivo antitumoral activity werepreserved.

A particularly preferred ErbB2-specific NK-92 cell or cell line of theinvention is the NK-92 cell or cell line identified as NK-92/5.28.zwhich was deposited on Jun. 11, 2014 at the Leibniz InstituteDSMZ—German Collection of Microorganisms and Cell Cultures GmbH,Inhoffenstr. 7B, 38124 Braunschweig, Germany, under accession number DSMACC3244.

The present invention demonstrates that it is feasible to engineerCAR-expressing NK cells as a clonal, molecularly and functionallywell-defined and continuously expandable cell therapeutic agent, andsuggest NK-92/5.28.z cells as a promising candidate for clinicaldevelopment.

Medical Uses

As described above, the present invention provides the ErbB2-specificNK-92 cell or cell line according to the invention for use in medicine.

As described above, the present invention provides the NK-92 cell orcell line according to the invention for use in the prevention and/ortreatment of cancer, preferably ErbB2-expressing cancers.

Preferably, the cancer, preferably the ErbB2-expressing cancers, is/areselected from

-   -   breast cancer,    -   ovarian cancer,    -   gastric cancer,    -   prostate cancer,    -   squamous cell carcinoma,    -   head and neck cancer,    -   colon cancer,    -   pancreatic cancer,    -   uterine cancer,    -   renal cell cancer,    -   glioblastoma,    -   medulloblastoma,    -   sarcoma    -   lung cancer.

In a preferred embodiment, the cells are pre-treated by irradiation,preferably by γ-irradiation.

Said irradiation may be included as a safety measure.

As described above, the present invention provides the NK-92 cell orcell line according to the invention for use as targeted celltherapeutic agent and/or for adoptive cancer immunotherapy.

A “cell therapeutic agent”, in particular a “targeted cell therapeuticagent” or “targeted allogeneic cell therapeutic agent” refers to animmune cell suitable for application for adoptive cancer immunotherapy,which is genetically modified to express an antigen receptor thatspecifically recognizes a defined antigen expressed on the surface of atarget tumor cell.

“Adoptive, target-cell specific immunotherapy” or “adoptive cancerimmunotherapy” or “adoptive cell therapy (ACT)” refers to a form oftherapy in which immune cells are transferred to tumor-bearing hosts.The immune cells have antitumor reactivity and can mediate direct orindirect antitumor effects.

Genetic engineering of NK-92 cells with CARs, such as provided by thisinvention, is very suitable for ACT and the treatment of cancer.

Preferably, the use comprises a pre-treatment of the cells byirradiation, preferably γ-irradiation.

Said irradiation may be included as a safety measure.

Method for Generating an ErbB2-Specific NK-92 Cell or Cell Line

As described above, the present invention provides a method forgenerating an ErbB2-specific NK-92 cell or cell line.

Said methods further allows to molecularly identify the ErbB2-specificNK-92 cell or cell line generated.

Said method comprises the steps of:

(1) providing a vector for transducing NK-92 cells,

(2) transducing NK-92 cells with said vector,

(3) deriving/generating single cell clones by limiting dilution;

(4) identifying CAR-expressing cells by flow cytometric analysis withErbB2-Fc fusion protein,

(5) selecting cell clone(s) which display high and stable CAR-expressionduring continuous culture,

(6) evaluating cytotoxic activity of the retargeted cells againstErbB2-expressing cells,

(7) evaluating cytotoxic activity of the retargeted cells againstErbB2-negative cells,

(8) selecting cell clone(s) which display high cytotoxicity againstErbB2-expressing cells and low or no cytotoxicity against ErbB2-negativecells, and

(9) determining number and position of vector integration, and selectingthe cell clones exhibiting vector intergration in an intergenic regionon chromosome 2 and in the TRAF2 gene on chromosome 9.

The vector for transducing NK-92 cells of step (1) is an integratingvector, preferably a lentiviral vector.

An example for a suitable lentiviral vector is lentiviral transferplasmid pHR'SIN-cPPT-WPREmut vector (Schambach et al., 2006).

Further suitable lentiviral vectors are known to the skilled person.

The lentiviral vector encodes a chimeric antigen receptor (CAR)comprising

-   -   an ErbB2-specific scFv antibody fragment,    -   a hinge region,    -   transmembrane and intracellular domains of CD28,    -   and intracellular domain of CD3 zeta.

The lentiviral vector can be generated following established methodologyknown to a skilled person.

A suitable example for an ErbB2-specific scFv antibody fragment is scFvFRP5.

For example, the ErbB2-specific scFv antibody fragment comprises orconsists of the amino acid sequence of SEQ ID NO. 13 (scFv FRP5) and/oris encoded by the nucleotide sequence of SEQ ID NO. 14.

ErbB2-specific scFv FRP5 is further described in EP 2 164 516 B1 andU.S. Pat. No. 7,887,801 B2.

Chimeric antigen receptors (CARs) are known in the art. TheErbB2-specific CAR used in the present invention comprises:

(i) an ErbB2-specific scFv antibody fragment,

(ii) a hinge region,

(iii) transmembrane and intracellular domains of CD28, and intracellulardomain of CD3 zeta.

In one embodiment, the chimeric antigen receptor (CAR) comprises orconsists of the amino acid sequence of SEQ ID NO. 15 and/or is encodedby the nucleotide sequence of SEQ ID NO. 16.

Suitable examples for chimeric antigen receptors (CARs) comprising anErbB2-specific scFv antibody fragment are further known, such asdescribed in WO 2012/031744.

Step (1) preferably comprises producing VSV-G pseudotyped lentiviralvector particles.

Step (2) is preferably carried out by following GMP-compliantprocedures.

The NK-92 cells of step (2) are preferably human NK-92 cells.

Step (5) preferably comprises selecting cell clone(s) which display highand stable CAR-expression during continuous culture.

Steps (6) and (7) comprise in vitro cytotoxicity assays, such as FACSbased assays, preferably carried out at different effector to targetratios (E/T).

Suitable ErbB2-expressing cells are ErbB2-expressing tumor cells, suchas murine renal cell carcinoma cells stably expressing human ErbB2(Renca-lacZ/ErbB2) or MDA-MB453 human breast carcinoma cells (ATCC no.HTB-131).

Said ErbB2-negative cells are used for evaluating or testing thespecific cytotoxicity of the cell clones.

Suitable ErbB2-negative cells are NK-sensitive but ErbB2-negative cells,such as human K562 erythroleukemia cells (ATCC no. CCL-243).

Said ErbB2-negative cells are used for evaluating or testing the naturalcytotoxicity/endogenous, CAR-independent cytotoxicity of the cellclones.

The cytotoxicity assays in step (6) and/or (7) are preferably alsocarried out with unmodified NK-92 cells (ATCC no. CRL-2407) and theresults are compared with the results of the tested cell clones.

In step (8), the cell clone(s) are selected which display highcytotoxicity against ErbB2-expressing cells and low or no cytotoxicityagainst ErbB2-negative cells.

Step (9) preferably comprises determining number and position of vectorintegration by linear amplification-mediated PCR (LAM-PCR) and DNAsequencing, and confirmation by PCR analysis of genomic DNA.

Suitable primers for the PCR analysis of genomic DNA and theirrespective amplification products are, for example (in an embodimentwith a lentiviral vector):

Vector integration site: CAR vector integration in TRAF2 gene:

Amplification Forward Primer Reverse Primer Product 5′ part TRAF2-F1:CAR-R1: TRAF2-CAR (5′) CTTCAGCAGGGACCAGAAACAA CCGCTTAATACTGACGCTCTCG587 bp [SEQ ID NO. 1] [SEQ ID NO. 2] [SEQ ID NO. 9] 3′ part CAR-F1:TRAF2-R1: CAR-TRAF2 (3′) ATCGCCACGGCAGAACTCA GACCCTTCACCCAACGCTTAG503 bp [SEQ ID NO. 3] [SEQ ID NO. 4] [SEQ ID NO. 10]Vector integration site: CAR vector integration in intergenic region of chromosome 2:Amplification Forward Primer Reverse Primer Product 5′ part IGCHR2-F1:CAR-R2: IGCHR2-CAR TCAGTGGAATGGGCAGCTTCAAGT TTCAGCAAGCCGAGTCCTGCGT (5′)[SEQ ID NO. 5] [SEQ ID NO. 6] 679 bp [SEQ ID NO. 11] 3′ part CAR-F2:IGCHR2_CAR-R1: IGCHR2-CAR ACTGATAATTCCGTGGTGTTGT CACTGTGGCTCACTGCTAGA(3′) [SEQ ID NO. 7] [SEQ ID NO. 8] 376 [SEQ ID NO. 12]

As described above, the present invention provides an NK-92 cell or cellline obtained by the method according to the invention.

Method for Identifying an ErbB2-Specific NK-92 Cell or Cell Line

As described above, the present invention provides a method foridentifying an ErbB2-specific NK-92 cell or cell line.

Said method comprises the steps of:

-   -   determining number and position of vector integration in cell        (clones), and selecting the cell (clones) exhibiting vector        integration in an intergenic region on chromosome 2 and in the        TRAF2 gene on chromosome 9.

As discussed above, the method preferably comprises determining numberand position of vector integration by linear amplification-mediated PCR(LAM-PCR) and DNA sequencing, and confirmation by PCR analysis ofgenomic DNA.

Suitable primers for the PCR analysis of genomic DNA and theirrespective amplification products are, for example (in an embodimentwith a lentiviral vector), as described above.

As described herein above, preferably, the cell (clone) is characterizedin that by PCR analysis of the genomic DNA of said cell (clone) at leastone of the following amplification products is obtained:

-   -   PCR with primers of SEQ ID NOs. 1 and 2 yields an amplification        product with the nucleotide sequence of SEQ ID NO. 9;    -   PCR with primers of SEQ ID NOs. 3 and 4 yields an amplification        product with the nucleotide sequence of SEQ ID NO. 10;    -   PCR with primers of SEQ ID NOs. 5 and 6 yields an amplification        product with the nucleotide sequence of SEQ ID NO. 11;    -   PCR with primers of SEQ ID NOs. 7 and 8 yields an amplification        product with the nucleotide sequence of SEQ ID NO. 12.

As described above, the present invention provides an NK-92 cell or cellline identified by the method according to the invention.

As described above, the present invention provides the NK-92 cell orcell line obtained or identified by the methods according to theinvention for use in the prevention and/or treatment of cancer,preferably ErbB2-expressing cancers.

Preferably, the cancer, preferably the ErbB2-expressing cancers, is/areselected from:

-   -   breast cancer,    -   ovarian cancer,    -   gastric cancer,    -   prostate cancer,    -   squamous cell carcinoma,    -   head and neck cancer,    -   colon cancer,    -   pancreatic cancer,    -   uterine cancer,    -   renal cell cancer,    -   glioblastoma,    -   medulloblastoma,    -   sarcoma, and    -   lung cancer.

In a preferred embodiment, the cells are pre-treated by irradiation,preferably by γ-irradiation.

Said irradiation may be included as a safety measure.

As described above, the present invention provides the NK-92 cell orcell line obtained or identified by the methods according to theinvention for use as targeted (allogeneic) cell therapeutic agent and/orfor adoptive cancer immunotherapy.

Preferably, the use comprises a pre-treatment of the cells byirradiation, preferably γ-irradiation.

Said irradiation may be included as a safety measure.

Methods of Prevention and/or Treatment

According to the present invention this object is solved by a method forthe prevention and/or treatment of cancer (herein after “treatmentmethod”).

The cancer is preferably the ErbB2-expressing cancers, which is/arepreferably selected from:

-   -   breast cancer,    -   ovarian cancer,    -   gastric cancer,    -   prostate cancer,    -   squamous cell carcinoma,    -   head and neck cancer,    -   colon cancer,    -   pancreatic cancer,    -   uterine cancer,    -   renal cell cancer,    -   glioblastoma,    -   medulloblastoma,    -   sarcoma, and    -   lung cancer.

Preferably, said treatment method comprises or includes adoptive cancerimmunotherapy.

Said treatment method comprises

-   -   administering to a subject in a therapeutically effective amount    -   (a) NK-92 cells according to the present invention or NK-92        cells obtained by the method according to the present invention,        and    -   (b) optionally, respective excipient(s).

A “therapeutically effective amount” of the NK-92 cells of thisinvention refers to the amount that is sufficient to treat therespective disease (cancer) or achieve the respective outcome of theadoptive, target-cell specific immunotherapy.

PREFERRED EMBODIMENT

According to the present invention, CAR-engineered NK-92 cells weredeveloped as a targeted allogeneic cell therapeutic agent. Here, wedescribe the generation and the molecular and functionalcharacterization of a clonal ErbB2-specific NK-92 cell line suitable forclinical applications. These NK-92/5.28.z cells were derived from asingle cell clone after lentiviral transduction with a vector encoding asecond generation CAR that targets the ErbB2 (HER2) receptor tyrosinekinase, a tumor-associated self-antigen expressed at elevated levels bymany human cancers of epithelial origin (Hynes and Lane, 2005).

Following GMP-compliant procedures, we generated an NK-92/5.28.z singlecell clone expressing an ErbB2-specific CAR with CD28 and CD3ζ signalingdomains. Vector integrations were mapped by linearamplification-mediated PCR and DNA sequencing. In vivo tumor homing andantimetastatic activity were evaluated in NOD-SCID IL2Rγ^(null) (NSG)mouse models.

ErbB2-specific NK-92/5.28.z cells efficiently lysed ErbB2-expressingtumor cells in vitro that were resistant to unmodified NK-92 cells.Importantly, specific recognition of ErbB2-positive tumor cells andantitumoral activity were retained in vivo, resulting in homing ofNK-92/5.28.z cells to orthotopic breast carcinoma xenografts andreduction of pulmonary metastasis of renal cell carcinoma cells inmurine models.

The data shown in the present application demonstrate successfultargeting of human NK-92 cells to the tumor-associated cell surfaceantigen ErbB2 by expression of a humanized and codon-optimizedsecond-generation CAR. Following GMP-compliant procedures, weestablished from a molecularly defined single cell clone a continuouslyexpanding CAR-modified cell line suitable for clinical development.These NK-92/5.28.z cells display stable CAR expression upon prolongedculture and target-antigen-specific cytotoxicity in vitro and in vivo.The possibility to fully characterize this cell clone at the molecularand cellular levels adds an important degree of safety for the clinicalapplication of the clone, in contrast to the heterogeneous compositionof CAR-modified primary NK and T cells, in which no exhaustive molecularanalysis is possible.

In mice fluorochrome-labeled NK-92/5.28.z cells selectively enriched inErbB2-positive orthotopic breast carcinoma xenografts within 24 hoursafter intravenous injection, while unmodified NK-92 cells failed toaccumulate in tumors. This demonstrates that NK-92/5.28.z cells retaintarget cell specificity in vivo, and are capable of penetrating tissuesand homing to distant tumor sites. In an experimental metastasis modelbased on ErbB2-expressing renal cell carcinoma cells, intravenousinjection of NK-92/5.28.z cells reduced metastasis formation indifferent experiments by 50% or more, while unmodified NK-92 cellsfailed to affect outgrowth of pulmonary tumor nodules. Importantly, invivo antitumor activity of NK-92/5.28.z cells irradiated with 10 Gy wasthe same as that of non-irradiated cells. This may be relevant forfuture clinical application of NK-92/5.28.z, where irradiation of cellsmay be included as a safety measure as previously done in phase Iclinical trials with unmodified NK-92 cells (Arai et al. 2008; Tonn etal., 2013).

Immune cells in tumor patients are often functionally compromised due tothe immunosuppressive activity of the cancer. Hence, for adoptive cancerimmunotherapy with NK cells, donor-derived allogeneic cells are beingpreferred since they do not recognize tumor cells as ‘self’, therebybypassing inhibitory signals (Geller and Miller, 2011). We have shown,that this advantage can be extended to CAR-engineered NK-92 cells.

Our data demonstrate that it is feasible to develop CAR-engineered NK-92cells in a similar manner as a clonal, molecularly and functionallywell-defined and continuously expandable off-the-shelf cell therapeuticagent with selective and markedly enhanced antitumor activity in vitroand in vivo. Such cells are clinically useful for the treatment ofvarious ErbB2-positive malignancies. Thereby the potent antitumoractivity, the immediate availability as a fully characterizable cellproduct, and the lack of obvious risks of manufacturing failures makesthese cells a valid and cost-effective alternative to CAR-modifiedpatient T cells.

The following examples and drawings illustrate the present inventionwithout, however, limiting the same thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show chimeric antigen receptor expression and selectivecytotoxicity of clonal NK-92/5.28.z cells.

FIG. 1A) is a schematic representation of lentiviral transfer plasmidpS-5.28.z-W encoding under the transcriptional control of the SpleenFocus Forming Virus promoter (SFFV) the chimeric antigen receptor (CAR)5.28.z. CAR 5.28.z consists of an immunoglobulin heavy chain signalpeptide (SP), the ErbB2-specific scFv (FRP5) antibody fragment (scFv), ahinge region (CD8α), followed by transmembrane and intracellular domainsof CD28 and the intracellular domain of CD3ζ. The CAR-encoding sequenceis flanked by 5′ and 3′ long terminal repeats (LTR) of the vector (notshown).

FIG. 1B) is a graph showing CAR-expression by the clonal NK-92/5.28.zcell line generated under GMP conditions by transduction with lentiviralvector S-5.28.z-W determined by flow cytometry with ErbB2-Fc fusionprotein (open area). Unmodified NK-92 cells served as control (grayarea).

FIG. 1C) are graphs showing specific cell killing by NK-92/5.28.z cells(filled circles) in FACS-based cytotoxicity assays at different effectorto target ratios (E/T) using murine renal cell carcinoma cells astargets that stably express human ErbB2 (Renca-lacZ/ErbB2) or human EGFR(Renca-lacZ/EGFR) as a control. Unmodified NK-92 cells were included forcomparison (open circles).

FIG. 1D) is a graph showing natural cytotoxicity of NK-92/5.28.z cellsagainst NK-sensitive but ErbB2-negative targets compared to unmodifiedNK-92 cells using human K562 erythroleukemia cells as targets.

FIGS. 2A, 2B and 2C are graphs showing molecular characterization ofclonal NK-92/5.28.z cells.

FIG. 2A) is a schematic representation of the integration sites of thelentiviral CAR vector S-5.28.z-W (shaded box) flanked by 5′ and 3′ longterminal repeat (LTR) sequences (filled boxes) in genomic DNA (openboxes). The PCR strategy to map the integration sites in the TRAF2 geneon chromosome 9 and in an intergenic region on chromosome 2 isindicated.

FIGS. 2B and 2C are images showing PCR analysis of vector integrations.

FIG. 2B) shows specific DNA sequences encompassing the junctions betweenthe TRAF2 gene and the 5′ end of the integrated CAR vector (TRAF2-CAR),and between the 3′ end of the integrated CAR vector and the TRAF2 gene(CAR-TRAF2) amplified by PCR with genomic DNA from 3 different passagesof NK-92/5.28.z cells as template and the indicated oligonucleotideprimer pairs, yielding characteristic 587 and 503 bp amplificationproducts. Genomic DNA of unmodified NK-92 cells as well as sampleswithout template DNA (H₂O) were included as controls.

For FIG. 2C), likewise, specific DNA sequences encompassing thejunctions between the intergenic region in chromosome 2 and 5′(IGCHR2-CAR) and 3′ ends (CAR-IGCHR2) of the integrated CAR vector wereamplified, yielding characteristic 679 and 376 bp amplificationproducts.

M: DNA marker (GeneRuler 100 bp Plus DNA Ladder, Thermo Scientific).

FIGS. 3A and 3B are graphs showing growth and cytotoxic activity ofNK-92/5.28.z cells upon γ-irradiation.

For FIG. 3A), to investigate the effect on viability, NK-92/5.28.z cellswere irradiated with 5 or 10 Gy and cultured for up to 72 hours.Proliferation was analyzed by counting viable cells at the indicatedtime points using trypan blue exclusion.

FIG. 3B) shows cytotoxic activity of NK-92/5.28.z cells 24 hours afterirradiation with 10 Gy against ErbB2-positive MDA-MB453 andErbB2-negative MDA-MB468 breast carcinoma cells determined in FACS-basedcytotoxicity assays at different effector to target ratios (E/T) asindicated (filled circles). Unmodified NK-92 cells 24 hours afterirradiation were included for comparison (open circles).

FIG. 4. Homing of NK-92/5.28.z cells to ErbB2-positive breast carcinomasin vivo.

Unmodified NK-92 (upper panel) or ErbB2-specific NK-92/5.28.z cells(lower panel) were labeled with fluorescent DiD labeling reagent andintravenously injected into NSG mice carrying established orthotopicMDA-MB453/EGFP breast carcinoma xenografts. Twenty-four hours afterinjection, tumors were excised, single cell suspensions were prepared,and analyzed for the presence of EGFP-expressing and DiD-labeled cells.DiD-positive NK cells are indicated in dark grey (lower rightquadrants). EGFP-positive breast carcinoma cells (upper left quadrants)and double-negative murine stromal cells (lower left quadrants) areindicated in light grey. Double-positive events (upper right quadrant)represent conjugates of CAR-expressing NK-92/5.28.z and MDA-MB453/EGFPtarget cells. Representative flow cytometric data from one animal ofeach group are shown (n=3).

FIGS. 5A and 5B are graphs showing in vivo antitumor activity ofNK-92/5.28.z cells.

For FIG. 5A), to investigate antitumor activity, NSG mice wereintravenously injected with Renca-lacZ/ErbB2 renal cell carcinoma cells.Then animals were treated twice by i.v. injection of unmodified NK-92 orclonal NK-92/5.28.z cells at days 1 and 3 after tumor cell injection.

Control mice received PBS. Four weeks after tumor challenge, lungs wereexcised and tumor nodules on the lung surface were counted.

For FIG. 5B), in a separate experiment, NSG mice injected withRenca-lacZ/ErbB2 cells were treated as described above withnon-irradiated NK-92 or NK-92/5.28.z cells, or NK-92/5.28.z cellsirradiated with 10 Gy as indicated. Mean values±SEM are shown; n=5. ns,p>0.05; *, p<0.05; **, p<0.01.

EXAMPLES Example 1

1.1 Methods

Cells and Culture Conditions

Human K562 erythroleukemia cells (ATCC, Manassas, Va.) were maintainedin RPMI 1640 medium (Lonza, Köln, Germany). Human MDA-MB453 andMDA-MB468 breast carcinoma cells, and HEK 293T cells (all ATCC) werecultured in DMEM (Lonza). All media were supplemented with 10%heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin. Human NK-92 cells (ATCC) were propagated in X-VIVO 10medium (Lonza) supplemented with 5% heat-inactivated human plasma(German Red Cross Blood Service Baden-Württemberg—Hessen, Frankfurt,Germany) and 100 IU/ml IL-2 (Proleukin; Novartis Pharma, Nurnberg,Germany). Murine Renca-lacZ/ErbB2 and Renca-lacZ/EGFR renal cellcarcinoma cells expressing human ErbB2 or EGFR were cultured inRPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mlpenicillin, 100 μg/ml streptomycin, 0.25 mg/ml zeocin and 0.48 mg/mlG418 (Maurer-Gebhard et al., 1998).

Generation of CAR-Expressing NK-92/5.28.z Cells

The CAR sequence 5.28.z was designed by in silico assembly of animmunoglobulin heavy chain signal peptide, ErbB2-specific scFv(FRP5)antibody fragment and a modified CD8α hinge region (wherein an unpairedcysteine within the hinge region was replaced by serine), followed byCD28 transmembrane and intracellular domains and CD3ζ intracellulardomain. A codon-optimized fusion gene was synthesized (GeneArt,Regensburg, Germany) and inserted into lentiviral transfer plasmidpHR'SIN-cPPT-WPREmut vector (Schambach et al., 2006), resulting inlentiviral transfer plasmid pS-5.28.z-W. VSV-G pseudotyped vectorparticles were generated and NK-92 cells were transduced as describedpreviously (Sahm et al., 2012), and for cell clone NK-92/5.28.zconfirmed by PCR analysis of genomic DNA with oligonucleotide primersthat yielded characteristic PCR products spanning the junctions betweenchromosomal DNA and integrated vector sequences.

Cytotoxicity Assays

Cytotoxicity of NK-92 cells towards target cells was analyzed inFACS-based assays as described (Sahm et al., 2012). Briefly, targetcells were labeled with calcein violet AM (Molecular Probes, Invitrogen,Karlsruhe, Germany) and co-cultured with effector cells at variouseffector to target (E/T) ratios for 2 h at 37° C. After co-culture, 250μl of a 1 μg/ml propidium iodide (PI) solution were added to each sample5 min before flow cytometric analysis in a FACSCanto II flow cytometer(BD Biosciences, Heidelberg, Germany). Data were analyzed using FACSDivasoftware (BD Biosciences). To calculate specific cytotoxicity, thenumber of spontaneously lysed target cells in the absence of effectorcells was subtracted from the number of dead target cells determined ascalcein violet AM and PI double positive in the measured sample.

Irradiation of NK-92 Cells

NK-92/5.28.z and unmodified NK-92 cells were collected bycentrifugation, counted, washed, resuspended in fresh growth medium andirradiated with 5 or 10 Gy using a Biobeam 2000 device (Gamma ServiceMedical, Leipzig, Germany). For in vitro proliferation and cytotoxicityassays, irradiated cells were washed, resuspended in fresh growth mediumand cultured for up to 72 h. Proliferation was analyzed by countingviable cells at different time points using trypan blue exclusion. Forin vivo experiments, cells were irradiated with 10 Gy and applieddirectly.

Tumor Homing of NK-92 Cells

EGFP-expressing MDA-MB453 breast carcinoma cells were derived bytransduction of MDA-MB453 cells with an EGFP-encoding lentiviral vectorand enrichment by flow cytometric cell sorting. Orthotopic breastcarcinoma xenografts were induced in 4 to 6 weeks old female NOD-SCIDIL2R γ^(null) (NSG) mice (Charles River, Sulzfeld, Germany) by injectionof 5×10⁶ MDA-MB453/EGFP cells suspended in Matrigel (BD Biosciences)into the mammary fat pad. When tumors were palpable, NK-92/5.28.z orunmodified NK-92 cells were labeled with DiD (1,1′-dioctadecyl-3,3,3′,3′tetramethylindodicarbocyanine) labeling reagent (Molecular Probes/LifeTechnologies, Darmstadt, Germany) as described (Tavri et al., 2009), andinjected into the lateral tail vein of the tumor bearing mice (1×10⁷cells/animal; 3 animals per group). Twenty-four hours after injection,mice were sacrificed, tumors were excised, single cell suspensions wereprepared, and analyzed for the presence of EGFP-expressing andDiD-labeled cells in a FACSCanto II flow cytometer.

In Vivo Antitumor Activity

Four to 6 weeks old female NSG mice were injected with 1×10⁵Renca-lacZ/ErbB2 cells into the lateral tail vein at day 0. Then animalswere treated by i.v. injection of 1×10⁷ NK-92/5.28.z or unmodified NK-92cells at days 1 and 3 after tumor cell injection (5 mice/group). Controlmice received PBS. In separate experiments, NSG mice injected withRenca-lacZ/ErbB2 cells were also treated with irradiated NK-92/5.28.zcells (10 Gy), or non-irradiated NK-92/5.28.z and unmodified NK-92 cellsas controls (5 mice/group). Four weeks after tumor challenge, allanimals were sacrificed, lungs were excised, and tumor nodules on thelung surface were counted as described (Maurer-Gebhard et al., 1998).

Statistical Analysis

Differences between values were evaluated using the two-tailed unpairedStudent's t test. P values <0.05 were considered significant.Statistical calculations were performed using Prism 5 software (GraphPadSoftware, La Jolla, Calif.).

1.2 Results

Generation of an ErbB2-Specific NK-92/5.28.z Single Cell Clone

The chimeric antigen receptor 5.28.z was used to generate a clinicallyapplicable ErbB2-specific NK-92 cell line (FIG. 1A). VSV-G pseudotypedlentiviral CAR vector particles were produced and NK-92 cells from acertified NK-92 master cell bank (Arai et al., 2008) were transduced.Single cell clones were derived by limiting dilution, and CAR-expressingcells were identified by flow cytometric analysis with ErbB2-Fc fusionprotein. A total of 15 CAR-expressing single cell clones werefunctionally and molecularly characterized, which harbored between oneand four vector copies. One cell clone termed NK-92/5.28.z whichdisplayed high and stable CAR-expression during continuous culture in asetting reflecting large-scale expansion under GMP conditions wasselected for further analysis (FIG. 1B). Selective cytotoxic activity ofthe retargeted cells was evaluated using Renca-lacZ/ErbB2 murine renalcell carcinoma cells stably expressing human ErbB2. Clonal NK-92/5.28.zcells displayed high cytotoxicity towards these ErbB2-expressing targetcells, which were resistant to unmodified NK-92 (FIG. 1C, left panel).In contrast, ErbB2-negative negative but otherwise isogenicRenca-lacZ/EGFR cells expressing epidermal growth factor receptordisplayed no enhanced sensitivity to the effector cells (FIG. 1C, rightpanel). This indicates that cell killing was indeed mediated byinteraction of CAR 5.28.z with its target antigen ErbB2.

Endogenous, CAR-independent cytotoxicity of NK-92/5.28.z cells wasinvestigated using ErbB2-negative but NK-sensitive K562 humanerythroleukemia cells as targets. While K562 cells were highly sensitiveto CAR-negative unmodified NK-92 cells, they were killed to a much lowerextent by ErbB2-specific NK-92/5.28.z cells (FIG. 1D).

Taken together, these data demonstrate that NK-92/5.28.z cells arehighly selective and efficiently kill ErbB2-expressing tumor cells,while their endogenous cytotoxicity to ErbB2-negative targets ismarkedly reduced when compared to unmodified NK-92 cells.

Molecular Characterization of ErbB2-Specific NK-92/5.28.z Cells

Linear amplification-mediated PCR (LAM-PCR) and DNA sequencing revealedone vector integration each in an intergenic region on chromosome 2, andin the TRAF2 gene on chromosome 9 of clonal NK-92/5.28.z cells (FIG.2A). The integration sites were confirmed by PCR analysis of genomic DNAof NK-92/5.28.z cells from three different passages during continuousculture over several months, thereby amplifying specific DNA sequencesthat encompass the junctions between the TRAF2 gene and the 5′ end ofthe integrated CAR vector, and between the 3′ end of the integrated CARvector and the TRAF2 gene (FIG. 2B, upper panels), as well as specificDNA sequences that encompass the junctions between the intergenic regionin chromosome 2 and 5′ and 3′ ends of the integrated CAR vector (FIG.2B, lower panels). In each case, genomic DNA of the different passagesof NK-92/5.28.z cells yielded the same characteristic amplificationproducts of defined length and sequence, demonstrating long-termstability of the vector integrations. No amplification products wereobtained with the same oligonucleotide primers upon PCR analysis ofgenomic DNA from unmodified NK-92 cells, indicating that specific PCRanalysis of the CAR vector integrations also represents a powerfuldiagnostic tool to molecularly identify the NK-92/5.28.z cell clone.

a) Amplification product TRAF2-CAR (5′)

CAR vector integration in TRAF2 gene

5′ part of vector integration

PCR product TRAF2-CAR (5′) from genomic DNA of NK-92/5-28.z cells

Primers:

TRAF2-F1: [SEQ ID NO. 1] CTTCAGCAGGGACCAGAAACAA CAR-R1: [SEQ ID NO. 2]CCGCTTAATACTGACGCTCTCGlower case letters: TRAF2 geneupper case letters: vector sequenceLength: 587 nucleotides

SEQ ID NO. 9 cttcagcagggaccagaaacaaaactcacactctttcttctctgagttga  50gactggaaaaatgaaagattgttttaggggaaacttgagggaacagtctg 100ggcagcctgcagggcatggccctgttcctccagggctgggaaagtcagca 150ctgctttctggtggcgaACTGGAAGGGCTAATTCACTCCCAACGAAGACA 200AGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAG 250CCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAA 300AGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTC 350TGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTA 400GCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGC 450TCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAG 500GGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAA 550GGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGG 587b) Amplification product CAR-TRAF2 (3′)CAR vector integration in TRAF2 gene3′ part of vector integrationPCR product CAR-TRAF2 (3′) from genomic DNA of NK-92/5-28.z cellsPrimers:

CAR-F1: [SEQ ID NO. 3] ATCGCCACGGCAGAACTCA TRAF2-R1: [SEQ ID NO. 4]GACCCTTCACCCAACGCTTAGlower case letters: TRAF2 geneupper case letters: vector sequenceLength: 503 nucleotides

SEQ ID NO. 10 ATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGG  50GGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGAATTCGATAC 100TCGAGGTCGAGGCAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACA 150AGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAA 200GGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGG 250GTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAG 300GGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTA 350GTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACC 400CTTTTAGTCAGTGTGGAAAATCTCTAGCAccttccctctgcagctgctgg 450ctcagccgattgtatatgctgggagctctgcactaagcgttgggtgaagg 500 gtc 503c) Amplification product IGCHR2-CAR (5′)CAR vector integration in intergenic region of chromosome 25′ part of vector integrationPCR product IGCHR2-CAR (5′) from genomic DNA of NK-92/5-28.z cellsPrimers:

IGCHR2-F1: [SEQ ID NO. 5] TCAGTGGAATGGGCAGCTTCAAGT CAR-R2:[SEQ ID NO. 6] TTCAGCAAGCCGAGTCCTGCGTlower case letters: intergenic region chromosome 2upper case letters: vector sequenceLength: 679 nucleotides

SEQ ID NO. 11 tcagtggaatgggcagcttcaagttgatgtcatttcaatagtaacttatt  50tcagtctacatacttcccaagaatgcaccatctcttttttatgtatttat 100tattttgagaaagagtctcactctgtcgcccaggctggagtgcaatggca 150tgatcttggctcactgtaacctccgtctcctgggttcaagccattctcct 200gtctcagcctcccgggtagtggggttataggcacacaccaccacgcccgg 250ctaatttttgtatttttagtaaagatggggtttcaccatgttggccaggc 300tgggctcaaactcttgacttcaggtgatccgcccaccttggcctcccaaa 350gtgctgggatgacaggcACTGGAAGGGCTAATTCACTCCCAACGAAGACA 400AGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAG 450CCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAA 500AGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTC 550TGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTA 600GCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGC 650TCTCTCGACGCAGGACTCGGCTTGCTGAA 679d) Amplification product CAR-IGCHR2 (3′)CAR vector integration in intergenic region of chromosome 23′ part of vector integrationPCR product CAR-IGCHR2 (3′) from genomic DNA of NK-92/5-28.z cellsPrimers:

CAR-F2: [SEQ ID NO. 7] ACTGATAATTCCGTGGTGTTGT IGCHR2_CAR-R1:[SEQ ID NO. 8] CACTGTGGCTCACTGCTAGAlower case letters: intergenic region chromosome 2upper case letters: vector sequenceLength: 376 nucleotides

SEQ ID NO. 12 ACTGATAATTCCGTGGTGTTGTCGAATTCGATACTCGAGGTCGAGGCAAT  50TCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCT 100TAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCC 150AACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGAC 200CAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAA 250GCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGT 300TGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGG 350AAAATCTCTAGCAgtgagccacagtg 376Target Cell Killing by Irradiated NK-92/5.28.z Cells

In phase I clinical trials with unmodified NK-92, irradiation of cellswith 10 Gy prior to infusion had been included as a safety measure toprevent permanent engraftment (Arai et al., 2008; Tonn et al., 2013).Similar safety measures may be important for clinical use of retargetedNK-92 cells. Hence, we tested the effects of γ-irradiation on growth andcytotoxic activity of clonal NK-92/5.28.z cells. Irradiation with 5 Gywas sufficient to prevent further replication, while the number ofviable NK-92/5.28.z cells remained almost constant for 24 hours afterexposure to 5 or 10 Gy before declining gradually (FIG. 3A). To assesseffects on cytotoxic activity, NK-92/5.28.z cells irradiated with 10 Gywere cultured for 24 hours and then co-incubated for two hours withErbB2-expressing human MDA-MB453 breast carcinoma cells as targets.Irradiated NK-92/5.28.z displayed high and specific cytotoxicity towardsMDA-MB453 cells (65% specific lysis at an E/T ratio of 10:1), while theydid not lyse ErbB2-negative human MDA-MB468 breast carcinoma cells (FIG.3B). Neither MDA-MB453 nor MDA-MB468 cells were killed by irradiatedunmodified NK-92 cells.

Homing of NK-92/5.28.z Cells to ErbB2-Positive Breast Carcinomas

The potential of NK-92/5.28.z cells to home to established tumors wasinvestigated in an orthotopic breast carcinoma model. MDA-MB453 cellstransduced with an EGFP-encoding lentiviral vector were implanted intothe mammary fat pad of female NSG mice, and allowed to grow until tumorswere palpable. Then NK-92/5.28.z and unmodified NK-92 cells were labeledwith fluorescent DiD labeling reagent, and intravenously injected intothe tumor-bearing animals. Twenty-four hours later, tumors were excised,single cell suspensions were prepared, and analyzed for the presence ofEGFP-expressing tumor cells and DiD-labeled NK cells. In mice injectedwith unmodified NK-92, only a few of the NK cells were found in thetumors (FIG. 4, upper panel). In contrast, NK-92/5.28.z cells werestrongly enriched in MDA-MB453/EGFP xenografts (FIG. 4, lower panel).Importantly, we also found conjugates of NK-92/5.28.z and MDA-MB453/EGFPcells in the cell suspensions prepared from the tumors. These datademonstrate that NK-92/5.28.z cells retain target cell specificity invivo, and are capable of penetrating tissues and homing to distant tumorsites.

In Vivo Antitumor Activity of NK-92/5.28.z Cells

For evaluation of in vivo antitumor activity we chose an experimentallung metastasis model. NSG mice received intravenous injections ofRenca-lacZ/ErbB2 cells, followed by i.v. injections of unmodified NK-92or retargeted NK-92/5.28.z cells at days 1 and 3 after tumor cellinoculation. Control mice received PBS. Four weeks after tumorchallenge, lungs were excised and tumor nodules on the lung surface werecounted. While treatment with unmodified NK-92 cells did not affectmetastasis formation in comparison to PBS-treated controls, retargetedNK-92/5.28.z cells reduced the number of pulmonary tumor nodules in thisexperiment by approximately 50% (mean number of lung surface metastases:PBS: 37.7±5.4; NK-92: 36±5.1; NK-92/5.28.z: 19±2.6; p<0.05) (FIG. 5A).To assess whether NK-92/5.28.z cells retain in vivo antitumor activityafter γ-irradiation, a similar experiment was performed employingNK-92/5.28.z cells that were irradiated with 10 Gy prior to injection.Control animals were treated with non-irradiated NK-92/5.28.z ornon-irradiated unmodified NK-92 cells. In comparison to treatment withunmodified NK-92 cells, both, non-irradiated and irradiated NK-92/5.28.zcells markedly reduced the number of pulmonary tumor nodules (meannumber of lung surface metastases: NK-92: 68.3±9.1; NK-92/5.28.z:21±4.9; irradiated NK-92/5.28.z: 14.4±5.5; p<0.01) (FIG. 5B). These datademonstrate specific antitumor activity of systemically appliedNK-92/5.28.z cells against ErbB2-expressing tumor cells in a modelreflecting disseminated disease. Importantly, viability andfunctionality of NK-92/5.28.z cells were transiently preserved afterγ-irradiation at a dose that prevents further effector cell replication,permitting target cell recognition and killing in vivo.

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The invention claimed is:
 1. A method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of ErbB2-specific NK-92 cells, the cells containing a genomically integrated lentiviral vector encoding a chimeric antigen receptor comprising an ErbB2-specific scFv antibody fragment, a hinge region, transmembrane and intracellular domains of CD28, and intracellular domain of CD3 zeta, wherein the cells are NK-92/5.28.z cells having accession number DSM ACC3244, and wherein administration treats the cancer in the subject.
 2. The method of claim 1, wherein said vector is genomically integrated (i) in an intergenic region on chromosome 2, and (ii) in the TRAF2 gene on chromosome
 9. 3. The method of claim 1, wherein the cell or cell line is characterized in that by PCR analysis of the genomic DNA of said cell or cell line at least one of the following amplification products is obtained: PCR with primers of SEQ ID NOs. 1 and 2 yields an amplification product with the nucleotide sequence of SEQ ID NO. 9; PCR with primers of SEQ ID NOs. 3 and 4 yields an amplification product with the nucleotide sequence of SEQ ID NO. 10; PCR with primers of SEQ ID NOs. 5 and 6 yields an amplification product with the nucleotide sequence of SEQ ID NO. 11; PCR with primers of SEQ ID NOs. 7 and 8 yields an amplification product with the nucleotide sequence of SEQ ID NO.
 12. 4. The method of claim 1, wherein the cells show reduced or no natural cytotoxicity to ErbB2-negative cells.
 5. The method of claim 1, wherein the cells show increased cytotoxicity to ErbB2-expressing tumor cells compared to unmodified NK-92 cells.
 6. The method of claim 1, wherein the ErbB2-specific scFv antibody fragment comprises the amino acid sequence of SEQ ID NO.
 13. 7. The method of claim 1, wherein the ErbB2-specific scFv antibody fragment is encoded by the nucleotide sequence of SEQ ID NO.
 14. 8. The method of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO.
 15. 9. The method of claim 1, wherein the chimeric antigen receptor is encoded by the nucleotide sequence of SEQ ID NO.
 16. 10. The method of claim 1, wherein the cancer is an ErbB2-expressing cancer.
 11. The method of claim 1, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, gastric cancer, prostate cancer, squamous cell carcinoma, head and neck cancer, colon cancer, pancreatic cancer, uterine cancer, renal cell cancer, glioblastoma, medulloblastoma, sarcoma, and lung cancer.
 12. The method of claim 1, wherein the method comprises pre-treatment of the cells by irradiation.
 13. The method of claim 1, wherein 1×10⁵ to 1×10⁷ cells are administered to the subject.
 14. The method of claim 1, wherein the cells are administered intravenously.
 15. The method of claim 12, wherein the irradiation is γ-irradiation. 